Expression Of Schlafen2 Gene In Cells And Its Biological Effection

Background:Schlafen family genes which participates regulation of thoracic gland development were discovered by Schwarz DA in 1998.There are only six literatures published,so it is very few about them to know so far.In these results,embryo-fatal phaenotype-DDK syndrome would be caused when the family gene is defective.As a group of genes which were discovered newly,the role of Schlafen family genes in tumor formation and development is indefinite.Schlafenl(Slfn1)could lead cell cycle to end in G1 stage by resisting the derivation of Cyclin D1.It is not clear to know the Gene function of Slfn2 at present.There's no cell line which is established to express Slfn2 gene stably,which restricts the development of further researching.For this reason,it is a brand-new subject to explore the effects of Slfn2 gene.It will be bound to provide a fresh view angle for advancement of researching human genome.So we could carry out more polynary and systematical researches.Objective:(1)Slfn2 was transiently transfected into NIH/3T3 cells.To observe the position of Slfn2 gene directly with laser scanning confocal microscopy.Because the expressing proteins of Slfn2 were marked by green fluorescent protein(GFP).NIH/3T3 cell is a kind of mouse embryo-source blastoid cells.(2)To establish cell line which could express Schlafen2(Slfn2)gene stably and to elucidate biological functions of the gene.(3)To done the promoter region sequences of Slfn2.It will lay the foundation of prospective work.In future studies,the promoter domain and relative activity of Slfn2 gene in mice will be determined by some means,including to construct double report gene expressing vector and to detect the relative activity of luciferase.Methods:(1)To clone the whole coding region sequences of Slfn2 gene.(2)To construct pEGFPSlfn2 and pCI-Slfn2.neo eukaryotic expressing vectors.(3)pEGFP-Slfn2 vector and contrast control were transiently transfected into NIH/3T3 cells to observe the position of Slfn2 gene directly with laser scanning confocal microscopy.(4)pEGFP- Slfn2 and pCI-Slfn2.neo were stably transfected into NIH/3T3 cells respectively with Lipofectamine^TM2000,and G418 screening was used to obtain resistant cell strains.(5)The expression of Slfn2 was measured by Northern blot.(6) Growth curve and Transwell were utilized to analyze the effects of Slfn2 on cell proliferation and migration.(7)To clone the promoter region sequences of Slfn2 in order to connect the sequences to pGL3-Basic vector.The eukaryotic expressing vector which formed in the above-mentioned step will be cotransfect with pRL-CMV into NIH/3T3 cells.Therefore,we could determine the promoter domain and relative activity of Slfn2 gene in mice by detecting the relative activity of luciferase in future.Results:1.The whole coding region sequences of Slfn2 were gained by RT-PCR.The result of DNA sequencing proved that the products were consonant with the sequences of Slfn2 in Genebank.2.Restriction enzymes were used on the pEGFP-Slfn2 and pCI-Slfn2.neo eukaryotic expressing vectors.By means of this,we identified the vectors.Subsequently,the results of DNA sequencing told us that the Slfn2 genes were correct and competent for transfection.The genes were used in carrier conjugation.3.pEGFP-Slfn2 vector and contrast control were transiently transfected into NIH/3T3 cells to observe the position of Slfn2 gene directly with laser scanning confocal microscopy.There're green fluorescence in cell nucleus and cytolymph.The photos demonstrated that the proteins expressed by the gene were detected in cell nucleus and cytolymph.4.3T3 cells were stably transfected with the plasmid expressing Slfn2 and corresponding empty vectors.By screened with G418,we obtained resistant cell clones.The expression of Slfn2 was measured by Northern blot.Chose the positive cells and cultivated them to establish cell line.The results showed that the expressions of Slfn2 in these cell strains were correct and stable.5.Drawing growth curve of the cells which were transfected.The results showed that the cell proliferation was inhibited markedly when the expression of Slfn2 was stable in these cells. 6.Transwell experiment was used to analyze and detect the migration of negative controls and the cell clones stably expressing Slfn2.The results told us that the capability of migration was degrade strikingly when the expression of Slfn2 was stable in these cells.7.The promoter region sequences of Slfn2 were gained by PCR.The result of DNA sequencing showed that the sequences were identical with the one of Slfn2 in Genebank.Conclusions:(1)The eukaryotic expressing vectors containing Slfn2 were constructed successfully.Moreover,cell line which could express Slfn2 gene stably was established by first time. (2)Biological character of tumor cells would change obviously when Slfn2 expressed stably.It plays a part in nagtive regulation of cell proliferation and migration.Proliferation and migration were inhibited markedly when the expression of Slfn2 was stable in these cells.It is possible that Slfn2 is a potential tumor suppressor gene.Therefore,the expression of the gene is essential at tumor formation and development.(3)The promoter region sequences of Slfn2 were amplified successfully, which can be used to determine the promoter domain and relative activity of Slfn2 gene in mice in future.